1. Field of the Invention
The object of the present invention is novel preparations for a broad-spectrum antiplasmodial vaccine.
The object of the invention is also a vaccinating antigen of Plasmodium falciparum capable of inducing a resistance to the parasite which reproduces that observed in the mechanism of protective immunity or premunition.
The object of the invention is also preparations of monoclonal or polyclonal antibodies or chimeric fragments obtained from these antibodies specific for these antigens and likely to form part of a composition for passive immunotherapy.
Finally, the object of the invention is a kit permitting the in vitro diagnosis of the infection of an individual by a broad spectrum of plasmodial strains.
2. Description of the Related Art
The parasites responsible for malaria in man, including in particular Plasmodium falciparum or Plasmodium vivax to mention only the principal ones, exhibit different morphologies in the human host and express different antigens as a function of their localization in the organism of the infected host. The morphological and antigenic differences of these parasites during their life cycle in man enable at least four distinct stages of development to be defined.
The very first stage of development of the parasite in man corresponds to the sporozoite form introduced into the blood of the host by bites of insect vectors of the parasite. The second stage corresponds to the passage of the parasite into the liver and to the infection of the hepatic cells in which the parasites develop to form the hepatic schizonts which, when they are mature (for example in the case of P. falciparum on the 6th day after penetration of the sporozoites) release hepatic merozoites by bursting. The third stage is characterized by the infection of the blood erythrocytes by the asexual forms (merczoites) of the parasite, this erythrocytic stage of development corresponds to the pathogenic phase of the disease. The fourth stage corresponds to the formation of the forms with sexual potential (or gametocytes) which will become extracellular sexual forms or gametes in the mosquito.
It is known that very many studies have been undertaken to isolate from strains of parasites which infect a human host polypeptide fractions to permit the in vitro diagnosis of malaria by the detection of the corresponding antibodies, on the one hand, and to attempt to vaccinate against malaria, on the other.
In 1976 the maintenance (so long-awaited) of P. falciparum in continuous culture in human RBC was accomplished (Trager and Jensen, Science 1976, 193: 673; Haynes et al., 1976) Nature 263:767-769. This achievement facilitated access to the parasite considerably and stimulated research which since then has experienced a rapid development. Efforts have been oriented mainly towards the development of a vaccine which in future will be necessary to control malaria whose incidence is becoming worse in as much as resistance of the parasite to drugs is spreading in different parts of the world.
In the search for a vaccine against the agent responsible for malaria, biologists are confronted with various problems not observed with other infectious agents such as viruses or bacteria. Of these special difficulties with the parasite we will mention principally:
The complexity of the biological cycle of the plasmodium taking place in two different hosts, the mosquito and man, undergoing sexual reproduction in the one and 2 different phases of asexual multiplication in the other. Thus 2 stages take place in man differing in their site of development (the liver and the blood circulation) and in their antigenic specificities.
The antigenic diversity of the parasite. Since 1983 the plasmodial antigens have been cloned and their nucleotide and protein sequences have been analyzed. This detailed study shows that more than 50% of the known antigens exhibit a high degree of polymorphism from one strain to another.
At the immunological level, the host-parasite relationship is very subtle As has already been mentioned, for a given parasite it is very different depending an the host in which it evolves. This leads to the difficulty of interpretation of the results obtained in the experimental models.
Furthermore, in the natural infection sterilizing immunity is never seen like that observed for example in viroses. However, there is no doubt that an acquired immunity exists but it is partial and labile.
Thus the complexity and the diversity of the parasite as well as the unusual nature of the immune response that it elicits are the major reasons for the absence of an anti-malarial vaccine at present.
The research approach most often taken in the development of a vaccine against malaria due to P. falciparum hence consists of the identification (on the basis of the information cited above) of a potential candidate, and then the evaluation of its value either in vitro by testing the specific antibodies in the inhibition of the growth of the parasite or of certain of its properties (cytoadhesion, rosette formation . . . ), or in vivo by the immunization of monkeys often with the complete Freund adjuvant. The present situation may thus be summed up as the existence of a large number of potential candidates characterized by their biochemical properties, their nucleotide and protein sequences, their degree of polymorphism, their localization on the parasite etc. Nevertheless, the researchers dispose of limited means for assessing the value of their candidates: 1) in vitro tests implicating mechanisms of action of antibodies whose validity in vivo is poorly documented, 2) vaccinations of non-human primates, and hence the evaluation of the effect of a vaccine on an experimental infection is based on parasitological and clinical parameters and particularly the type of immunity which may be induced which are very different from those of the natural infection in man.
The strict specificity of the host-parasite relationship leads under natural conditions to the opposite of what is observed in the animal models, to an equilibrium in which the parasite survives by inducing in its natural host a non-sterilizing immunity. The chronic nature of the parasitic infection suggests that the majority of the molecular components of the parasite are selected so as to protect the micro-organism against the immune defences of the individual infected, and do so by means of escape which are very varied but specifically adapted to the natural host. In the experimental host, the poorly adapted parasite defends itself less well against the immune system and protection against a single treated infection is easy to obtain, and vaccination is still easier to obtain.
Gordon-Thomson, Immunity in Malaria, Trans. Roy.Soc. Trop. Med. Hyg. XXVI (6) 483-514) dearly concluded that immunity against P. falciparum can only be acquired in the regions where transmission is essentially continuous year after year. This "tolerance" to parasitism requires at the individual level an uninterrupted infection for about 15 years, sometimes 20 years and up to 26 years in a study conducted in Panama. An immunity associated with a latent infection necessary for the maintenance of the protection results from this. Sergent (1935) Institute Pasteurd Algerie Archives, 3:279 suggested the term "premunition" to define this "particular state of resistance contemporaneous with the infection and ceasing with it".
Thus the immunity (or premunition) against P. falciparum acquired by man in a holo- or hyperendemic zone is characterized by:
a very long delay prior to its installation (15 to 20 years of infection) PA1 its incapacity to abolish the infection, it is a non-sterilizing immunity. PA1 its lability. In the absence of any reinfection (during more than one year), the premunition is lost and the subject again becomes susceptible to the disease if subject to a new infection. PA1 the biochemical and immunological characterization of the 48 kD protein, PA1 the screening of a genomic library of the plasmodium for its capacity to inhibit the coupling of a specific monoclonal antibody of the IgM type which has the special characteristic of blocking the ADCI-type reaction ("antibody-dependent cellular inhibition") induced by the specific IgG of the plasma of the subjects protected by premunition PA1 the characterization of the proteins synthesized by the clones selected, PA1 the sequencing of the insert of the clone selected PA1 the search for the functional effect of the antibodies corresponding to this protein in the tests described.
The indications in favour of humoral immunity in acquired protection against malaria come from the first attempts at passive transfer of serum from an individual in the "chronic" phase who had reached a state of premunition (i.e. showing circulating parasites in small numbers without any clinical manifestation) to a subject in the acute phase. The condition of this latter is found to be improved subsequent to this passive transfer (Sotiriades 1917, Attempts at serotherapy in malaria Greek Med. XIX: 27-28)
The role of antibodies in premunition is demonstrated by several experiments of passive transfer carried out at the beginning of the 1960s. The transfer of IgG purified from hyperimmune African adult serum cures child victims of an acute infection by appreciably reducing their parasitemia (Cohen et al., 1971, Trans. Roy. Soc. Trop. Med. Hyg. 65 (2): 125-135; McGregor et al., 1964, the passive transfer of human material immunity, Am. J. Trop. Med Hyg. 13: 237-239). The newborn are protected up to the third month of their life as a result of maternal antibodies; this is proved by the beneficial effect of the IgG of the umbilical cord transferred to children suffering from an acute attack due to P. falciparum (Edozien et al., 1962) Lancet 2:951.
The development of immunity and its efficacy in the protection of man against P. falciparum nonetheless proves the existence of parasite molecules which are the targets of an effective immune defence.
Recent experiments have made it possible to show that a) the G immunoglobulins (IgG) of immune African adults are protective by passive transfer in man infected with malaria (Sabchareon et al., Amer. J. of Trap. Med. and Hyg., vol. 45, No.3, September 1991, 297-308), b) that, contrary to what is believed to be established, these antibodies are incapable of directly inhibiting the invasion of red cells by the parasites; on the other hand, they act by an antibody-dependent cellular inhibition mechanism (ADCI) in which the monocyte plays the role of effector cell (Bouharoun-Tayoun et al., J. Exp. Med., vol. 172, December 1990 pp. 1633-1641; S. KHusmith et al., 1983, Inf. Imm. 41 (1): 219 and F. Lunel et al., 1989 Inf. Imm. 57: 2043), c) This mechanism necessarily implicates cytophilic antibodies, i.e. those capable of binding to the monocyte through their Fc receptor, in fact, there has been observed in the serum of protected subjects a prevalence of cytophilic isotypes IgG1 and IgG3 and in non-protected subjects a preponderance of non-cytophilic classes, IgG2 and/or IgM (H. BOUHAROUN-TAYOUN et al., 1992, Infection and Immunity, pp. 1473-1481).